Calcium channels - An overview

Calcium Channels – An Overview

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C

Department of Neuroscience, NIS Center of Excellence, CNISM Research Unit, Torino, Italy

Definition

▶Voltage-gated Ca2±channelsare integral membrane proteins forming aqueous pores which open in response to cell depolarization. Ca2+ channels play a key role in controlling vital functions: they shape the ▶action potentialand membrane electrical oscillations and act as gate-controller of Ca2+_{, the most ubiquitous}

▶second messenger [1]. As such, Ca2+ channels are implicated in cardiac, skeletal and smooth ▶muscle contraction (▶excitation-contraction coupling),▶hormoneand neu-rotransmitter release (▶excitation-secretion coupling) and Ca2+_{-dependent processes that modulate short- and}

long-term cell activity and gene expression (▶excitation-transcription coupling) [2–5].

Characteristics

Ca2+ channels have been grouped into two main classes, based on their threshold of activation: the▶high voltage-activated (HVA) channelsand the▶low voltage-activated (LVA) channels[4]; although this classification could not be strictly applied since some of the HVA channels activate at significantly low voltages [2]. LVA channels activate “▶transiently” during small depolar-izations near▶resting membrane potentials(→ ▶Mem-brane potential – basics) and are therefore commonly indicated as T-type channels. T-type channels are res-ponsible for ▶low-threshold spikes, oscillatory cell activity, muscle contraction, hormone release, cell growth, differentiation and proliferation [5]. The HVA channels require larger membrane depolarizations to open and are further subdivided into four types (▶L-, N-, P/Q- and R-type Ca2±channels) based on their structural, pharmacological and biophysical characteristics [2,3]. They are responsible for the sustained depolarizing phase of action potentials, muscle contraction, hormone and neurotransmitter release, gene expression and cell differentiation.

Molecular Structure

The principal pore-forming subunit of both LVA and HVA channels is the α1-subunit, a high- molecular

weight protein (190–250 kDa), which is structurally similar to the▶Na±channelα-subunit [1]. It is formed by four domains (I–IV) linked together in a single polypeptide chain and each domain contains six putative transmembrane segments (S1–S6), plus a loop (P) that dips partially into the pore to form the pore-lining region (Fig. 1). The cytoplasmic loops

linking the four domains are structurally important for their interactions with β-subunits, second messengers, membrane binding proteins and channel▶gating.

Molecular cloning of Ca2+ _{channels has provided}

evidence for the existence of ten different pore-forming α1 subunits with pharmacological and biophysical

profiles similar to the endogenous Ca2+ channels expressed in most tissues. Alignment of their amino acid sequences suggests strong homologies and diver-gences between the various Ca2+_{channel types (Fig. 2).}

Strong homologies exist between the four L-types (Cav1), the N-, P/Q- and R-type (Cav2), and the three

T-type (Cav3) channels, while large divergences exist

between the HVA and LVA subfamilies. Figure 2 reports the Ca2+ _{channel classifications most used in}

the literature [2,3].

At variance with the T-type channels, whose α1-subunit is sufficient to warrant ▶activation and ▶inactivation gating, channel expression and mem-brane incorporation, the HVA channels are hetero-multimeric protein complexes which comprise the α1-subunit in association with auxiliary β, α2-δ and

γ-subunits (Fig. 3). Coexpression of α2-δ with the

α1-subunit ensures proper Ca2+current kinetics and

cur-rent densities. The same occurs with the co-expression of β and α1-subunits. Still vague is the role of γ-subunits

(γ1to γ8) which consist of four transmembrane domains

and whose site of interaction with the α1-subunit

is still unknown. The α2-δ-subunit is formed by a

membrane-spanning δ-peptide (27 kDa) and an extra-cellular α2-peptide (143 kDa) bound together by a

disulfide bridge (Fig. 1). Presently, four genes encoding for different α2-δ-subunits (α2-δ1to α2-δ4) have been

identified with several additional splice variants. The up-regulation of the α2-δ gene appears to correlate

with the onset of ▶allodynia (non-noxious stimuli eliciting pain) and ligands that target the α2-δ-subunit,

the gabapentins, are used for treatment of neuropathic pain. The four β-subunits (β1 to β4) and their splice

variants so far identified are almost exclusively cytosolic. They possess a hydrophobic region contain-ing SH3 and guanylate kinase domains, indicatcontain-ing that they belong to the membrane-associated guanylate kinase (MAGUK) family and as such may integrate multiple signaling pathways near the channel. The β-subunit has high-affinity for a conserved region within the domain I-II region of N- and PQ-type chan-nels, termed the alpha interaction domain (AID). Activation-Inactivation Gating

As for other voltage-gated ion channels, the probability of Ca2+_{channels opening is strictly voltage-dependent,}

i.e., the switch from a closed (non-conductive) to an open (conductive) configuration is strictly dependent on voltage, usually requiring 4–8 mV to change e-fold the probability of channel opening [6]. Structure-function

studies have associated this property to the presence of positively charged lysine and arginine residues distributed in the S4 transmembrane segment of each domain, which form the “▶voltage sensor” (Fig. 1). However, the exact location of the activation gates that

regulate the open and closed states of the pore remains unknown.

During maintained depolarization, Ca2+ channels tend to inactivate, but the speed and extent of channel inactivation may vary dramatically. In general, Ca2+

channels inactivate by either Ca2+_{- or voltage-dependent}

mechanisms. Ca2+ _{-dependent inactivation is}

domi-nant for the cardiac Cav1.2 L-type channel, but may also

occur for other HVA channels.▶Ca2+-dependent inacti-vation is a ▶calmodulin-dependent process, involving sites on the C-terminal domain of the α1-subunit.

Voltage-dependent inactivation is a general term for inactivation that does not clearly depend on Ca2+. ▶T-type Ca2+ channelstend to inactivate rapidly and almost completely, while inactivation of HVA channels is usually slow and incomplete. Spontaneous switch between inactivating and non-inactivating modes have been observed in single N-type channels, perhaps reflecting modulation by some intracellular signaling process. However, how Ca2+ -binding or membrane voltage affect the inactivation gate is still widely unknown [6].

Channel Permeability

The present view of Ca2+ _{channel permeability is}

based on the existence of an intrapore binding site controlling both ion selectivity and channel block: the▶selectivity filter. This highly specialized region of Ca2+channels consists of a ring of four negative charged groups inside the pore. In HVA channels, each of the four P loops contains a glutamate forming the EEEE locus, in T-type channels two glutamates are substituted by two aspartates in the corresponding position (EEDD locus). The spatial arrangement of the four negative charges

Calcium Channels – An Overview. Figure 1 Subunit structure of voltage-gated Ca2+_{channels: transmembrane}

topology of the α1and associated auxiliary subunits (β, α2-δ, γ). Predicted α-helices are depicted as cylinders.

For the α1-subunit the transmembrane spanning α-helices (1 to 6) are repeated in the four domains (I to IV). Red

cylinders indicate the positively charged S4 segments (voltage sensors), and the thick green lines denote the pore loops (P) that line the permeation pathway for Ca2+. The lengths of lines are not intended to represent the exact lengths of the polypeptide segments indicated. The same is for the size of various subunits. Adapted and redrawn from ref [2].

Calcium Channels – An Overview.

Figure 2 Phylogenetic tree of voltage-gated Ca2+ channels, showing the percentage of identity between the different cloned Ca2+_{channels [5]. The first}

bifurcation occurs between HVA and LVA channels. The HVA family includes four genes encoding the L-type channels (Cav1.1–Cav1.4) and three genes encoding

the P/Q (Cav2.1), N- (Cav2.2) and R-type (Cav2.3)

channels. The LVA family includes three genes encoding the Cav3.1–Cav3.3 channels. Adapted and redrawn

in the P loops is postulated to closely coordinate two Ca2+ _{ions whose sequential entrance and subsequent}

interaction induce high Ca2+ fluxes while preserving high affinity for the pore-site [7]. Although this simple ion-ion interaction would explain the dual nature of Ca2+_{as permeant ions at millimolar concentrations and}

as blockers of Na+ _{currents at micromolar}

concen-trations, the complete understanding of Ca2+ channel permeability is likely to require a further hypothesis of ion-pore structure interactions. The recent availability of T-type channel clones [5] has allowed closer comparisons between LVA and HVA channel permeability properties, highlighting the role that the EEEE or EEDD locus play in the regulation of ion selectivity in the two channel groups. Cav3.1 T-type channels have apparently a

nar-rower pore size (5.1 Å diameter) compared to the Cav1.2

L-type pore size (6.2 Å diameter) [7]. This structural difference may explain the different Ca2+_/Ba2+

selectivi-ty and blocking action of Cd2+ and Ni2+ of the two channel families (LVA and HVA): the L-, N- and P/Q-type channels being more permeable to Ba2+than Ca2+ and more sensitive to the block by Cd2+_{and the T-type}

channels being equally permeable to Ca2+_{and Ba}2+_and

more sensitive to the block by Ni2+_{(Table 1).}

Physiology, Pharmacology and Channelopathies In the following paragraphs are described the tissue and cellular location, physiological role, pharmacology and related ▶channelopathiesassociated to each Ca2+ channel type as summarized in Table 1.

L-type channels (Cav1).L-type channels are widely expressed in many tissues and control a number of Ca2+_{-dependent responses in excitable cells. In central}

neurons they are preferentially located on proximal dendrites and cell bodies and are involved in postsyn-aptic integration, neuronal plasticity, gene transcription and mood behavior. In sensory neurons (▶cochlear hair cells and photoreceptors), they directly control neurotransmitter release and sensory perception. The L-type family includes four α1-subunits (Cav1.1 to

Cav1.4) with different structure-function characteristics

but common blockers:▶1,4-dihydropyridines (DHPs), ▶phenylalkylamines and ▶benzothiazepines. With the exception of Cav1.3 and Cav1.4, which activate at

Calcium Channels – An Overview. Figure 3 Schematic representation of the voltage-gated Ca2+channel complex. On top is represented the heteromeric structure of the HVA Ca2+_{channel, consisting of the pore forming}

α1-subunit (with the four domains I to IV ) plus the β, γ and α2-δ auxiliary/regulatory subunits. To the bottom is

represented the LVA Ca2+channel consisting only of the pore forming α1-subunit. To the right are listed the different

α1-subunits that correspond to different Ca2+channel isoforms. Adapted and redrawn from ref [3].

relatively low voltages, the other Cav1 channels activate

at voltages much more positive than resting potential. Activation is fast and sharply voltage-dependent while inactivation is relatively slow in the presence of Ba2+ but speeds-up in the presence of Ca2+ (▶Ca2± -dependent inactivation). Deactivation (giving rise to ▶tail currents) is also fast, ensuring rapid closing of the channels on membrane repolarization to resting levels. L-type channels are distinguished from the other Cavchannels by their high sensitivity to DHPs. DHP

antagonists (nifedipine, nitrendipine) reversibly block the channels and help quantifying the amount of L-type

channels expressed in a cell, while DHP agonists (Bay K 8644) prolong the open state of the channel, producing slow tail currents near resting potential. As such, DHP agonists allow measuring the▶single channel activityof high conductance L-type channels in membrane patches, otherwise hardly detectable [8]. L-type channels can be often distinguished from the other Ca2+ _{channels for their}

▶cAMP/PKA-mediated up-regulationwhich causes increased mean open times and probability of openings at the single channel level and increased Ca2+ current amplitude in whole-cell recordings [9]. Neuronal and neuroendocrine L-type

Calcium Channels – An Overview. Table 1 Selective and often used blockers, distribution and established channelopathies [10] of the various voltage-gated Ca2+channels

Channel Blockers Distribution Channelopathies Selective Unselective (often used) Gene Diseases L Cav1.1 Dihydropyridines, phenylalkylamines, benzothiazepines

Cd2+ _{Skeletal muscles, transverse}

tubule

CACNA1S Hypokalemic periodic paralysis & malignant hyperthermia in humans, muscular dysgenesis in mice Cav1.2 Dihydropyridines, phenylalkylamines, benzothiazepines

Cd2+ Cardiac & smooth muscle myocytes; endocrine cells, neuronal cell bodies & dendrites

CACNA1C Timothy syndrome

Cav1.3 Dihydropyridines,

phenylalkylamines, benzothiazepines

Cd2+ Endocrine cells; neuronal cell bodies & dendrites, atrial myocytes & pacemaker cells, cochlear hair cells

CACNA1D Deafness, sinoatrial & atrioventricular node dysfunction Cav1.4 Dihydropyridines,

phenylalkylamines, benzothiazepines

Cd2+ Retinal rod & bipolar cells, spinal cord, adrenal gland, mast cells

CACNA1F Congenital stationary night blindness type 2, X-linked cone-rod dystrophy type 3 P/

Q

Cav2.1 ω-agatoxin IVA Cd2+ Nerve terminals & dendrites,

neuroendocrine cells

CACNA1A Episodic ataxia type-2, spinocerebellar ataxia type-6, familial hemiplegic migraine

N Cav2.2 ω-conotoxin VIA,

SNX 111 (ziconotide)

Cd2+ _{Nerve terminals & dendrites,}

neuroendocrine cells

CACNA1B

R Cav2.3 SNX 482 Cd2+, Ni2+ Nerve terminals & dendrites,

neuroendocrine cells, cardiac myocytes

CACNA1E

T Cav3.1 none Ni2+,

mibefradil

Neuronal cell bodies & dendrites, cardiac & smooth muscle myocytes

CACNA1G

Cav3.2 none Ni2+,

mibefradil

Neuronal cell bodies & dendrites, cardiac & smooth muscle myocytes,

neuroendocrine cells

CACNA1H Childhood absence epilepsy

Cav3.3 none Ni2+,

mibefradil

Neuronal cell bodies & dendrites

channels can be also inhibited by a fast ▶G-protein coupled receptor (GPCR) mechanism activated by neurotransmitters which could be at the basis of an ▶autocrine feedback controlof hormone release.

P/Q-type channels (Cav2.1). The Cav2.1 family includes two Ca2+channels which are nearly indistin-guishable except for their different affinity to a common blocker: the spider venom ω-agatoxin IVA. P-type chan-nels are more sensitive to ω-agatoxin IVA (Kd1–3 nM)

than Q-type channels (Kd100–200 nM). Cav2.1

chan-nels are widely expressed in neurons but are also available in pancreatic, pituitary and chromaffin cells. Their main physiological function is to control neuro-transmitter release in central neurons and mammalian ▶neuromuscular junctions where they are highly expressed at the presynaptic sites. P/Q-type channels control also the excitation-secretion coupling in pan-creatic and chromaffin cells. Cav2.1 channels play a key

role in neurotransmitter release due to their high-density of expression at the release sites of central synapses and share most of the modulatory properties of N-type channels described below. Similarly to the N-type channels they bind tightly to the ▶SNARE complex proteins at the▶synprint motifof the II-III linker of the channel. P/Q-type channels are also effectively inhib-ited by the neurotransmitter activated GPCRs mecha-nism described below. Missense mutations of P/Q-type channels cause ▶familial hemiplegic migraine (FHM) associated to an apparent gain of function as a result of an increased probability of channel openings and alteration of synaptic transmission (Table 1).

N-type channels (Cav2.2). Cav2.2 channels are

widely expressed in the central and peripheral nervous system and chromaffin cells. They are highly expressed at the nerve terminals, where they control neurotrans-mitter release, and to a minor degree at the dendritic sites, where they are involved in Ca2+_{signaling. Ca}

v2.2

channels control also hormone release in neuroendo-crine cells. The channels activate at relatively high voltages. Maximal activation at positive potentials and deactivation on return to resting levels are both fast. Inactivation is variable but significantly faster than L-type channels and slower than T-type channels. Cav2.2 channels are insensitive to DHPs but are

selectively blocked by ω-conotoxin GVIA and related cone snail toxins (Table 1).

N-type channels play a key role in neurotransmitter release due to their high-density of expression at the release sites and their tight binding to the SNARE complex of the vesicle release machinery (syntaxin 1A, SNAP-25, VAMP2/synaptobrevin and synaptotagmin). These proteins bind at the synprint motif of the II-III linker of the channel and the tight interaction affects the availability and gating of the channel. The SNARE complex in fact either steadily inactivates the channel or inhibits its activation through a Gβγ subunit.

Interestingly, N-type channels are effectively modulated by GPCRs activated by neurotransmitters and the mechanism is thought to be at the basis of▶presynaptic inhibition. Briefly, an activated Gβγsubunit binds directly

to the pore-forming α1-subunit of the N-type channel and

shifts the gating mode from “willing” (from which the channel readily opens) to “reluctant” (from which the channel opens less frequently). The ▶modulatory mechanismis▶membrane-delimited, voltage-dependent and causes an increased delay of the channel opening which produces an overall slow activation of the “reluctant” channel. Strong depolarizations opening the channels reduce the affinity of Gβγfor the α1-subunit and

the channel recovers its normal gating mode.

R-type channels (Cav2.3). Cav2.3 channels are

widely expressed in the central nervous system at the cell bodies, dendrites and presynaptic terminals. They are also expressed in ▶motoneurons, heart, pituitary and chromaffin cells. The Cav2.3 channel has been

originally reported to encode a Ca2+ _{channel type}

with biophysical properties between LVA and HVA channels, or usually as an HVA channel resistant to DHPs, ω-toxins and thus called R-type (for “residual”). Cav3 channels are likely to form a family of several

channels with fast activation but variable inactivation that could be fast and comparable to the Cav3 types or

slow like the Cav1 channels. They are involved in

neurotransmitter and hormone release, repetitive firing (→ ▶Action potential) and ▶long-term potentiation. The tarantula toxin SNX-482 blocks exogenously expressed Cav2.3 currents but is only partially or not

effective on native R-type currents, suggesting that Cav2.3 does not always conduct a significant portion of

the R-type current which remains after blocking all the other ▶voltage-gated Ca2+ channels. Cav2.3 channels

are also sensitive to small doses of Ni2+_{. In some case}

the Ni2+_{block has K}

dcomparable to that of the Cav3.2

T-type channel described below.

T-type channels (Cav3). Cav3 channels (Cav3.1 to

Cav3.3) are ubiquitously expressed and sustain key

physiological functions which derive from their unique properties [4,5]: (i) they activate and inactivate at unusually negative voltages and are responsible for a window-current near resting potentials, (ii) they exhibit fast and complete inactivation during sustained depolar-ization and deactivate slowly on repolardepolar-ization, (iii) they are equally or slightly more permeable to Ca2+_{than Ba}2+

and have small single channel conductance, (iv) they outlast ▶membrane-patch excision and (v) they are preferentially blocked by low doses of mibefradil and Ni2

+ _{(particularly the Ca}

v3.2) (Table 1). At present, Cav3

channels are recognized to play a critical role in many physiological functions in which a low-threshold Ca2+ entry is required to trigger, or sustain, specific cell activities. This is particularly true for the generation of low-threshold spikes, pacemaking activity, hormone

secretion, cell growth and differentiation. T-type channels play also a critical role in several pathologies in which either their recruitment, overexpression, or altered gating cause cardiac hypertrophy, hypertension, heart failure, ▶absence epilepsy,▶neurogenic pain, and▶Parkinson’s disease. Most recently, T-type channels are shown to control the vesicular release of neurotransmitters in neurons, and very recent data indicate that they are involved in fast ▶catecholamine release in adrenal chromaffin cells.

References

1. Hille B (2001) Ion channels of excitable membranes, 3rd edn. Sinauer, Sunderland, MA

2. Catterall WA, Perez-Reyez E, Snutch TP, Striessnig J (2005) International union of pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol Rev 57:411–425

3. Dolphin AC (2006) A short history of voltage-gated calcium channels. Br J Pharmacol 147:S56–S62 4. Carbone E, Lux HD (1984) A low voltage-activated, fully

inactivating Ca channel in vertebrate sensory neurones. Nature 310:501–502

5. Perez-Reyes E (2003) Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev 83:117–161

6. Jones SW (2003) Calcium channels: unanswered ques-tions. J Bioenerg Biomembr 35:461–475

7. Talavera K, Staes M, Janssens A, Klugbauer N, Droogmans G, Hofmann F, Nilius B (2001) Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca2+

channel α1G. J Biol Chem 276:45628–45635

8. Hess P, Lansman JB, Tsien RW (1984) Different modes of Ca channel gating behaviour favoured by dihydropyr-idine Ca agonists and antagonists. Nature 311:538–544 9. Bean BP, Nowycky MC, Tsien RW (1984) β-adrenergic

modulation of calcium channels in frog ventricular heart cells. Nature 307:371–375

10. Lorenzon NM, Beam K (2005) Calcium channelopa-thies. In: Zamponi G (ed) Voltage-Gated Calcium Channels. Kluwer Academic, London, UK, pp 240–261

Calcium Channels: Regulation of Gene

Transcription

A

S

, J. D

S

Synonyms

Excitation-transcription coupling; Voltage-gated calcium channels/ligand-gated calcium channels and control of gene expression; Calcium-regulated nuclear signaling

Definition

Nerve activity induces Ca2+_{influx through voltage-gated}

calcium channels (▶VGCCs), and activates vital func-tions such as▶neurotransmitter release(NT) and gene transcription. The latter function is often coupled to permanent changes to the structure of synapses (e.g., ▶synaptic plasticity) and the survivability of neurons. Recent research indicate that the specificity of local nuclear signaling pathways depends upon the association of specific channel types to cytosolic Ca2+-sensitive factors and not just Ca2+influx per se. Indeed, it is only the L-type (Cav1.2) channels which are critical for the

transcriptional regulation of many genes and these contribute only to a fraction of the total Ca2+ _influx

during neural activity. Ca2+ influx by other means, such as the▶N-methyl-D-aspartate receptors (NMDA), also contribute to calcium-dependent gene expression changes and share many but not all of the same nuclear signaling pathways.

Characteristics

Calcium as a Signaling Molecule

The nervous system relies on charged molecules to relay information. Whereas the relatively inert Na+and K+ _{ions serve mostly to establish membrane potential}

and carry action potentials along excitable membranes, Ca2+ _{has properties that allow it to accomplish}

additional functions. First, it has a unique affinity for binding ligands containing oxygen-donating groups such as carboxyls, carbonyls, ethers, and alcohols. Second, Ca2+_{is kept at low levels within the cytosol}

since it precipitates organic anions and is toxic to cells at high concentrations. These features likely lead to the exploitation of the calcium ion not only as a charge carrier but also as a signaling molecule.

Calcium-regulated Genes

Nerve activity promotes subcellular calcium “hotspots” or microdomains around individual channels, and collectively these active channels contribute to global cellular processes including synaptic plasticity and neuronal survival. An example is long-term memory which involves synaptic connectivity changes activated via ▶long term-potentiation (LTP). Strengthening of synapses induced by LTP requires gene transcription mediated by calcium-sensitive signaling pathways [1]. Furthermore, activity-dependent neuronal survival, which is important during cognitive development, also requires calcium-dependent gene transcription [2].

“First responder” genes are termed ▶immediate early genes (IEGs), whose transcription is upregulated without a requirement for newly synthesized proteins. The expression of IEGs is tightly regulated in space and time in active neurons by the summation of nerve inputs and local Ca2+ flux. ▶Brain-Derived Neurotrophic Factor (BDNF) is a classical calcium-activated IEG